Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems

Differential responses to opioids established the hypothesis that pre/postinspiratory (Pre-I) neurons of the parafacial respiratory group (pFRG) and inspiratory (Insp) neurons of the pre-Bötzinger complex (preBötC) constitute a dual brainstem respiratory center. For further analysis of pFRG/preBötC interactions, we studied in newborn rat brainstem-spinal cord preparations opioid and anoxia effects on histologically identified pFRG-driven “type-I” Insp preBötC neurons and Pre-I neurons from three distinct respiratory brainstem regions. The µ-opioid [d-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) slowed inspiratory-related cervical nerve bursts quantally, whereas anoxia induced nonquantal slowing and repetitive cervical bursts. DAMGO had no effect on membrane potential or input resistance of Pre-I neurons, while anoxia hyperpolarized them (~5 mV) and decreased their resistance (~30%). DAMGO prolonged the preinspiratory phase of Pre-I neuron bursting, whereas anoxia caused a shift to postinspiratory (48%) or inspiratory (22%) activity and silenced further 30% of cells. Pre-I neuron responses were not correlated with their rostrocaudal location or morphology. Neither DAMGO nor anoxia changed membrane potential of type-I neurons, but decreased their input resistance by 33% and 21%, respectively. The opposite DAMGO- and anoxia-evoked phase shifts of Pre-I neuron activity were reflected by corresponding shifts of pre/postinspiratory drive potentials in type-I neurons and, partly, by voltage-sensitive dye-imaged medullary neuronal population activities. The findings suggest that opioids presynaptically delay activation of type-I neurons as the target of drive from the pFRG to the preBötC. Contrary, anoxia seems to partly synchronize the pFRG and preBötC rhythm generators. This may enhance inspiratory and postinspiratory medullary activities for triggering multiple inspiratory motor bursts.     

Late-expiratory activity: emergence and interactions with the respiratory CpG

The respiratory rhythm and motor pattern are hypothesized to be generated by a brain stem respiratory network with a rhythmogenic core consisting of neural populations interacting within and between the pre-B?tzinger (pre-B?tC) and B?tzinger (B?tC) complexes and controlled by drives from other brain stem compartments. Our previous large-scale computational model reproduced the behavior of this network under many different conditions but did not consider neural oscillations that were proposed to emerge within the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) and drive preinspiratory (or late-expiratory, late-E) discharges in the abdominal motor output. Here we extend the analysis of our previously published data and consider new data on the generation of abdominal late-E activity as the basis for extending our computational model. The extended model incorporates an additional late-E population in RTN/pFRG, representing a source of late-E oscillatory activity. In the proposed model, under normal metabolic conditions, this RTN/pFRG oscillator is inhibited by B?tC/pre-B?tC circuits, and the late-E oscillations can be released by either hypercapnia-evoked activation of RTN/pFRG or by hypoxia-dependent suppression of RTN/pFRG inhibition by B?tC/pre-B?tC. The proposed interactions between B?tC/pre-B?tC and RTN/pFRG allow the model to reproduce several experimentally observed behaviors, including quantal acceleration of abdominal late-E oscillations with progressive hypercapnia and quantal slowing of phrenic activity with progressive suppression of pre-B?tC excitability, as well as to predict a release of late-E oscillations by disinhibition of RTN/pFRG under normal conditions. The extended model proposes mechanistic explanations for the emergence of RTN/pFRG oscillations and their interaction with the brain stem respiratory network.     

Postnatal developmental changes in activation profiles of the respiratory neuronal network in the rat ventral medulla

Two putative respiratory rhythm generators (RRGs), the para-facial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC), have been identified in the neonatal rodent brainstem. To elucidate their functional roles during the neonatal period, we evaluated developmental changes of these RRGs by optical imaging using a voltage-sensitive dye. Optical signals, recorded from the ventral medulla of brainstem–spinal cord preparations of neonatal (P0–P4) rats ( n = 44), were analysed by a cross correlation method. With development during the first few postnatal days, the respiratory-related activity in the pFRG reduced and shifted from a preinspiratory (P0–P1) to an inspiratory (P2–P4) pattern, whereas preBötC activity remained unchanged. The μ-opioid agonist [ d -Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) augmented preinspiratory activity in the pFRG, while the μ-opioid antagonist naloxone induced changes in spatiotemporal activation profiles that closely mimicked the developmental changes. These results are consistent with the recently proposed hypothesis by Janczewski and Feldman that the pFRG is activated to compensate for the depression of the preBötC by perinatal opiate surge. We conclude that significant reorganization of the respiratory neuronal network, characterized by a reduction of preinspiratory activity in the pFRG, occurs at P1–P2 in rats. The changes in spatiotemporal activation profiles of the pFRG neurones may reflect changes in the mode of coupling of the two respiratory rhythm generators.     

Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats

In brainstem-spinal cord preparations isolated from newborn rats, a whole cell recording technique was applied to record membrane potentials of inspiratory (Insp) and pre-inspiratory (Pre-I) neurons in the ventrolateral medulla. Labelling of these respiratory neurons with Lucifer Yellow allowed analysis of their locations and morphology. Intracellular membrane potentials from 25 Insp neurons were recorded. Average resting membrane potential was –49 mV (n=25) and input resistance was 306 M. Insp neurons were classified into three types from the patterns of synaptic potentials. Type I neurons (n=11) had a high probability of excitatory postsynaptic potentials (EPSPs) in the pre- and post-inspiratory phases. Type II neurons (n=7) showed abrupt transition to the burst phase from the resting potential level without increased EPSPs in the preinspiratory phase. Type III neurons (n=7) were hyperpolarized by inhibitory postsynaptic potentials (IPSPs) in the pre- and post-inspiratory phases. These Insp neurons, located in the ventrolateral medulla 80–490m from the ventral surface, were 10–30 m in diameter, and had various soma shapes (pyramidal, spherical or fusiform). Intracellular membrane potentials from 24 Pre-I neurons were recorded. The average resting membrane potential was –45 mV (n=24), and the input resistance was 320 M. Typical Pre-I neurons showed fairly great depolarization accompanied by action potentials during their burst phase and repolarization during the inspiratory phase. Most Pre-I neurons appeared to have a high level of synaptic activity. These cells were located in the ventrolateral medulla 50–440 m below the ventral surface and had pyramidal or fusiform somas of 10–25 m in diameter. Stimulation of the ipsilateral IXth, Xth roots or the spinal cord (C3 level) induced orthodromic responses in most Insp or Pre-I neurons. An antidromic action potential was induced in only one Pre-I neuron by stimulation at the ipsilateral C3 level. Many Insp or Pre-I neurons had dendrites that terminated close to the ventral surface of the medulla. The present study revealed postsynaptic activity of respiratory neurons in the rostral ventrolateral medulla, which is consistent with the excitatory and inhibitory synaptic connections from Pre-I neurons to Insp neurons, and inhibitory synaptic connections for Insp neurons to Pre-I neurons.     

Nociceptin/orphanin FQ causes non-quantal slowing of respiratory rhythm in brainstem-spinal cord preparation from newborn rat

Nociceptin/orphanin FQ (N/OFQ) is the endogenous agonist of the N/OFQ peptide receptor, an inhibitory G protein-coupled receptor. N/OFQ acts as a neuromodulator to depress respiratory rhythm in the brainstem. Although the mechanisms of respiratory rhythm generation remain poorly understood, the pre-inspiratory neuron (Pre-I) and the pre-Bötzinger complex (preBötC) inspiratory neuron (Insp) network in the rostral ventrolateral medulla (RVLM) have been proposed to be essential for respiratory rhythm generation. Opioids presumably cause quantal slowing via selective depression of preBötC Insps. However, it is unclear whether N/OFQ depresses respiratory rhythm via the same mechanism. In this study, using in vitro newborn rat en bloc preparations, we examined the slowing pattern of N/OFQ (quantal or non-quantal) and the effects of N/OFQ on the extracellularly recorded discharge of Pre-Is and Insps in the RVLM. N/OFQ caused non-quantal slowing with a synchronous decrease in burst rates of Insps and of C4 discharge whereas the intraburst spike number in Insps remained unchanged. It also caused a significant decrease in burst rates and intraburst spike numbers in Pre-Is, while the 1:1 coupling of Pre-Is bursts to C4 bursts was preserved. When superfusate K⁺ was elevated from 6.2 to 11.2 mM, Pre-I activity was increasingly uncoupled from C4 bursts. After the application of N/OFQ in a high [K⁺] superfusate, the 1:1 coupling of Pre-Is to C4 bursts was restored. We conclude that N/OFQ suppresses burst and spike generation of Pre-Is, and that suppression of Pre-Is activity with synchronous coupling to the Insps network contributes to N/OFQ-induced non-quantal slowing.     

Phox2b, RTN/pFRG neurons and respiratory rhythmogenesis

Phox2b-expressing cells in the parafacial region of the ventral medulla are proposed to play a role in central chemoreception and postnatal survival. Recent findings in the adult rat and neonatal mouse suggest that the Phox2b-immunoreactive (ir) cell cluster in the rostral ventrolateral medulla is composed of glutamatergic neurons and expresses neurokinin 1 receptor (NK1R), indicating that the cluster may be identical to the retrotrapezoid nucleus. This region overlaps at least partly with the parafacial respiratory group (pFRG) composed predominantly of pre-inspiratory (Pre-I) neurons that are involved in respiratory rhythm generation. Recently, we showed that Pre-I neurons in the parafacial region (pFRG/Pre-I) in neonatal rats are indeed expressing Phox2b and are postsynaptically CO₂ sensitive. Our findings suggest that Phox2b-expressing pFRG/Pre-I neurons play a role in respiratory rhythm generation as well as central chemoreception and thus are essential for postnatal survival. In this brief review, we focused on these recent findings and discuss the functional role of pFRG/Pre-I neurons.     

Modulation of respiratory rhythm by 5-HT in the brainstem-spinal cord preparation from newborn rat

 Effects of 5-hydroxytryptamine (5-HT) on inspiration-related nerve activity and membrane potential of respiratory neurons in the ventrolateral medulla were studied in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 5–100 μM 5-HT induced a biphasic response in inspiratory nerve activity: a transient increase in respiratory frequency followed by a decrease in the rate of discharge. The excitatory effect of 5-HT was particularly prominent in preparations with a respiratory rate of less than 3 min^–1, whereas the inhibitory effect was more pronounced in preparations with a higher respiratory rate. In pre-inspiratory (Pre-I) and inspiratory (Insp) neurons, 20 μM 5-HT induced a membrane depolarization of up to 10 mV accompanied by a significant decrease in the input resistance. Membrane depolarization by 5-HT was also evident in the presence of tetrodotoxin. In Pre-I neurons, 5-HT caused an increase in the burst rate, which was followed by a decrease in the intraburst firing frequency and burst amplitude, although the burst rate remained high. The burst rate in Insp neurons first increased and subsequently decreased without significant change in the intraburst firing frequency. Simultaneous intra- and extracellular recordings (in the contralateral medulla) of Pre-I/Pre-I neuron or Pre-I/Insp neuron pairs revealed that 5-HT disturbed the correlation between these neuron bursts. Increase in the respiratory rate induced by 20 μM 5-HT was completely blocked by pretreatment (5–15 min) with 5 μM ketanserin or 1 μM methysergide, but not by 10 μM propranolol. None of these antagonists blocked the inhibitory effects of 5-HT. A 5-HT₂ agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 10–100 μM) increased the respiratory rate. Perfusion with a 5-HT_1A agonist, 8-hydroxy-dipropylaminotetralin hydrobromide (8-OH-DPAT, 20–100 μM) induced an increase or a decrease in the respiratory rate. A 5-HT_2C agonist, 1-(3-chlorophenyl)piperazine (m-CPP, 2–10 μM) induced an initial decrease in the respiratory rate followed by a further long- lasting decrease. Burst activity of Pre-I neurons was suppressed upon administration of 10 μM m-CPP and enhanced with 20 μM DOI. The results suggest that changes in the bursting properties of Pre-I and Insp neurons induced by 5-HT lead to modulation of the respiratory network, thus causing biphasic modulation of the respiratory rhythm. In addition to effects via 5-HT_1A receptors, activation of 5-HT_2A and 5-HT_2C receptor subtypes might be involved in excitatory effects and inhibitory effects of 5-HT respectively. Received: 1 August 1997 / Received after revision: 27 October 1997 / Accepted: 4 November 1997     

Developmental changes in the spatio-temporal pattern of respiratory neuron activity in the medulla of late fetal rat

We investigated how the spatio-temporal pattern of respiratory neuron network activity in the ventral medulla changes during the late fetal period of rat. Brainstem-spinal cord preparations isolated from rat fetuses on embryonic days 17–21 (E17–E21) were stained with a voltage-sensitive dye for optical image analysis of neuronal activity of the ventral medulla. The spatio-temporal pattern of respiratory neuron activity in the preparation from E20 to E21 was basically identical to that of neonatal rat; pre-inspiratory activity in a limited region of the rostral ventrolateral medulla, the para-facial region, preceded by several hundred milliseconds the onset of inspiratory activity in the more caudal ventrolateral medulla, the pre-Bötzinger complex level. In contrast, in E17–E18 specimens, pre-inspiratory activity could not be detected in the rostral medulla at the level of the facial nucleus. Neuronal activity appeared to begin at the pre-Bötzinger complex level shortly before onset of the inspiratory burst. Strong activity then developed in the facial nucleus and peaked in the post-inspiratory phase. The transition of these patterns of respiratory activity occurred at E19. We conclude that the changes in the spatio-temporal pattern of neuronal activity reflect developmental changes in the cellular elements underlying rhythm generation in the fetal respiratory neuron network. We suggest that the pre-inspiratory neuron network of the para-facial region in the rostral ventrolateral medulla functions as the rhythm generator after E19/20.     

Localization of respiratory rhythm-generating neurons in the medulla of brainstem-spinal cord preparations from newborn rats

We describe the location of Pre-I neurons, which are important to respiratory rhythm generation, in the rostral medulla of brainstem-spinal cord preparations isolated from newborn rats. This neuronal group was delimited in the reticular formation slightly medial to the caudal area of the facial nucleus and near the ventral surface. The effects of electrical stimulation and lesions in that region were also examined with respect to respiratory rhythm generation. Single shock stimulation induced Pre-I neuron firing and reset the phase of the respiratory rhythm. Electrolytic lesions in the Pre-I neuron region reduced the respiratory rate.     

Maintenance of sympathetic tone by a nickel chloride-sensitive mechanism in the rostral ventrolateral medulla of the adult rat

In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection of the divalent cation nickel chloride (Ni(2+); 50 mM, 50 nl) into the rostral ventrolateral medulla elicited a dramatic inhibition of splanchnic sympathetic nerve activity (-44+/-6%) and a marked depressor response (-35+/-7 mmHg). Selective blockade of high-voltage activated Ca(2+) channels with omega-agatoxin IVA (P/Q-type), omega-conotoxin GVIA (N-type) and nifedipine (L-type) did not decrease arterial pressure or splanchnic sympathetic nerve activity when injected separately into the rostral ventrolateral medulla, or combined with kynurenate. Injection of caesium chloride or ZD 7288, a blocker of the hyperpolarization-activated cation current, into the rostral ventrolateral medulla had no effect on arterial pressure or splanchnic sympathetic nerve activity. Bilateral microinjection of nickel chloride into the caudal ventrolateral medulla/pre-B?tzinger complex elicited small increases in splanchnic sympathetic nerve activity (+17+/-13%) and arterial pressure (+12+/-4 mmHg). These were substantially smaller than those evoked by blockade of glutamatergic receptors or high-voltage activated Ca(2+) channels in this area. Injection of kynurenate or high-voltage activated Ca(2+) channel blocker, but not Ni(2+), in this area evoked respiratory termination. The results indicate the existence of a distinct mechanism maintaining the tonic activity of rostral ventrolateral medulla presympathetic neurons that is different from that maintaining the tonic activity in the caudal ventrolateral medulla/pre-B?tzinger region. We conclude that ion channels that are sensitive to Ni(2+), but are insensitive to high-voltage activated (L, P/Q, N) Ca(2+) channel blockers, and are located postsynaptically on the presympathetic rostral ventrolateral medulla neurons are responsible for the tonic activity of the presympathetic neurons in rostral ventrolateral medulla. These channels could well be the low-voltage-activated (or T-type) Ca(2+) channels although other conductances cannot be conclusively excluded.     

Modulation of expiratory motor output evoked by chemical activation of pre-Bötzinger complex in vivo

We have previously demonstrated that chemical stimulation of the pre-B?tzinger complex (pre-B?tC) in the anesthetized cat produces either phasic or tonic excitation of phrenic nerve discharge. This region is characterized by a mixture of inspiratory-modulated, expiratory-modulated, and phase-spanning (including pre-inspiratory (pre-I)) neurons; however, its influence on expiratory motor output is unknown. We, therefore, examined the effects of chemical stimulation of the pre-B?tC on expiratory motor output recorded from the caudal iliohypogastric (lumbar, L(2)) nerve. We found that unilateral microinjection of DL-homocysteic acid (DLH; 10 mM; 10-20 nl) into 16 sites in the pre-B?tC enhanced lumbar nerve discharge, including changes in timing and patterning similar to those previously reported for phrenic motor output. Both increased peak amplitude and frequency of phasic lumbar bursts as well as tonic excitation of lumbar motor activity were observed. In some cases, evoked phasic lumbar nerve activity was synchronized in phase with phrenic nerve discharge. These findings demonstrate that chemical stimulation of the pre-B?tC not only excites inspiratory motor activity but also excites expiratory motor output, suggesting a role for the pre-B?tC in generation and modulation of inspiratory and expiratory rhythm and pattern.     

Variations in motor patterns during fictive rostral scratching in the turtle: knee-related deletions

Agonist motor neurons usually alternate between activity and quiescence during normal rhythmic behavior; antagonist motor neurons are usually active during agonist motor neuron quiescence. During an antagonist deletion, a naturally occurring motor-pattern variation, there is no antagonist activity and no quiescence between successive bursts of agonist activity. Motor neuron recordings of normal fictive rostral scratching in the turtle displayed rhythmic alternation between activity and quiescence for hip flexors, knee flexors, and knee extensors. Knee-flexor activity occurred during knee-extensor quiescence. During a hip-extensor deletion, a variation of rostral scratching, rhythmic hip-flexor bursts occurred without intervening hip-flexor quiescence. There were 3 distinct patterns of knee motor activity during the cycle before or after a hip-extensor deletion. In most cycles, there was knee flexor-extensor rhythmic alternation. In some cycles, termed knee-flexor deletions, there was no knee-flexor activity and rhythmic knee-extensor bursts occurred without intervening knee-extensor quiescence. In other cycles, termed knee-extensor deletions, there was no knee-extensor activity and rhythmic knee-flexor bursts occurred without intervening knee-flexor quiescence. The concept of a module refers to a population of motor neurons and interneurons with similar activity patterns; interneurons in a module coordinate agonist and antagonist motor neuron activities, either with excitation of agonist motor neurons and interneurons, or with inhibition of antagonist motor neurons and interneurons. Previous studies of hip-extensor deletions support the concept of a rhythmogenic hip-flexor module. The knee-related deletions described here support the concept of rhythmogenic knee-flexor and knee-extensor modules linked by reciprocal inhibition.     

Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro

Summary It has previously been demonstrated that Pre-I neurons, localized in the rostral ventrolateral medulla, are important in the generation of the primary respiratory rhythm in brainstemspinal cord preparations from newborn rats. To investigate whether or not Pre-I neurons have endogenous pacemaker properties, we examined Pre-I neuron activity before and after chemical synaptic transmission was blocked by incubation in a low Ca²⁺ (0.2 mM), high Mg²⁺ (5 mM) solution (referred to here as low Ca). After incubation for about 30 min in low Ca, 28 (52%, type-1) out of 54 neurons tested in 27 preparations retained apparent rhythmic (phasic) activity after complete disappearance of C4 inspiratory activity. Sixteen neurons (30%, type-2) fired tonically and 10 (18%, type-3) were silent. We examined the effects of synaptic blockade on 14 inspiratory neurons in the RVL. The firing of all 14 neurons in 9 preparations disappeared concomitantly with the disappearance of C4 activity in low Ca. When the pH of the low Ca solution was lowered with a decrease in NaHCO₃ concentration from 7.4 to 7.1, the firing rate of the Pre-I neurons (type-1) increased from 12 to 18/min. In conclusion, the generator of respiratory rhythm in the newborn rat is probably a neuronal network with chemical synapses that functions mainly through the endogenous Pre-I pacemaker cells. Intrinsic chemoreception in the rhythm generator is probably important in frequency control of respiratory rhythm.     

Correlation analysis of respiratory neuron activity in ventrolateral medulla of brainstem-spinal cord preparation isolated from newborn rat

Cross correlation analysis was used to study functional connections between one inspiratory (I) neuron and another, and between one preinspiratory (Pre-I) neuron and another, in 54 brainstemspinal cord preparations isolated from newborn rats. Pre-I neurons usually fired in the pre and post inspiratory phases. Neurons were recorded extracellularly with pairs of microelectrodes placed on the same or opposite sides of the brainstem. Fourteen pairs of Pre-I neurons recorded bilaterally in the rostral ventrolateral medulla (RVL), 14 pairs of ipsilateral Pre-I neurons in the RVL, 14 pairs of bilateral I neurons in the RVL and 12 pairs of ipsilateral I neurons in the ventrolateral medulla were studied. Cross correlation histograms (CCHs) were computed. Significantly high peak bin counts were detected in 24 of 54 pairs. Peaks on one side of the origin of the CCHs were observed for one pair of ipsilateral Pre-I neurons, four pairs of bilateral I neurons and five pairs of ipsilateral I neurons. These findings suggest mono or oligo synaptic excitatory connections between paired neurons or shared inputs. Only one trough suggesting an oligo-synaptic inhibitory connection was evident in a CCH obtained from the pair of bilateral I neurons. This CCH revealed the peak and the trough on opposite sides of the origin, which was consistent with reciprocal excitatory and inhibitory connections between recorded neurons. Peaks on both sides of the origin were observed for three pairs of bilateral I neurons. From autocorrelation analysis and the latencies of these peaks, two of the three CCHs were consistent with reciprocal excitatory connections between recorded neurons, whereas the other CCH suggests shared inputs. Peaks at the origin were observed for two pairs of ipsilateral Pre-I neurons, four pairs of bilateral I neurons and five pairs of ipsilateral I neurons. These results suggest shared inputs. For Pre-I neurons recorded in opposite sides, no significant bin counts were detected. Peaks on one side were detected for three pairs. Present results suggest short term synchronisation of I neurons, and of Pre-I neurons via excitatory coupling, and the likelihood of comparatively strong interaction between I neurons, which may be important in maintaining the I burst.     

Defective interaction between dual oscillators for respiratory rhythm generation in Na+,K+-ATPase α2 subunit-deficient mice

The current concept regarding the respiratory centre in mammals is that it is composed of two distinct rhythm-generating neuronal networks in the ventrolateral medulla. These two rhythm generators can be active independently but are normally coupled in newborn and juvenile rats. Detailed characteristics of each generator and the neuronal mechanisms of coupling during development remain to be elucidated. Here, we report a knockout mouse (Na⁺,K⁺-ATPase α2 subunit gene ( Atp1a2 ) knockout) that may be defective in functional coupling between the two respiration-related rhythm generators. We investigated respiration-related neuron activity in an en bloc brainstem–spinal cord preparation isolated from embryonic day 18.5 Atp1a2^{−/ −} mouse fetuses. In the presence of adrenaline, two different types of rhythm generators were identified. One produced inspiratory burst activity that correlated with C4 inspiratory activity and was thought to be the inspiratory rhythm generator on the basis of its location and sensitivity to a μ-opiate receptor agonist, [ d -Ala2, N -Me-Phe4, Gly5-ol]-enkephalin (DAMGO). The other was presumed to be the preinspiratory rhythm generator because it was insensitive to DAMGO and correlated with facial nerve activity. Coupling between these rhythm generators did not function in the normal manner in Atp1a2^{−/ −} mice, as shown by disruption of the linkage between the preinspiratory burst and the inspiratory burst. Coupling was partially restored by repeated activation of the neurons within the networks, suggesting the involvement of an activity-dependent process in the prenatal development of this coupling.     

Glutamate neurotransmission is not required for, but may modulate, hypoxic sensitivity of pre-Bötzinger complex in vivo

Focal hypoxia in the pre-B?tzinger complex (pre-B?tC) in vivo elicits excitation of inspiratory motor output by modifying the patterning and timing of phrenic bursts. Hypoxia, however, has been reported to enhance glutamate release in some regions of the brain, including the medullary ventral respiratory column; thus the pre-B?tC-mediated hypoxic respiratory excitation may result from, or be influenced by, hypoxia-induced activation of ionotropic glutamate [i.e., excitatory amino acid (EAA)] receptors. To test this possibility, the effects of focal pre-B?tC hypoxia [induced by sodium cyanide (NaCN)] were examined before and after blockade of ionotropic EAA receptors [using kynurenic acid (KYN)] in this region in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Before blockade of ionotropic EAA receptors, unilateral microinjection of NaCN (1 mM; 10-20 nl) into the pre-B?tC produced either phasic or tonic excitation of phrenic nerve discharge. Unilateral microinjection of KYN (50-100 mM; 40 nl) decreased the amplitude and frequency of basal phrenic nerve discharge; however, subsequent microinjection of NaCN, but not DL-homocysteic acid (DLH, a glutamate analog), still produced excitation of phrenic motor output. Under these conditions, the NaCN-induced excitation included frequency modulation (FM) of phasic phrenic bursts, and in many cases, augmented and/or fractionated phrenic bursts. These findings show that the hypoxia-sensing function of the in vivo pre-B?tC, which produces excitation of phrenic nerve discharge, is not dependent on activation of ionotropic glutamate receptors, but ionotropic glutamate receptor activation may modify the expression of the focal hypoxia-induced response. Thus these findings provide additional support to the concept of intrinsic hypoxic sensitivity of the pre-B?tC.     

Effects of graded focal cold block in rostral areas of the medulla

Unilateral focal cold blocks (20 degrees C) in structures located ventrolaterally in rostral medulla consistently caused apnoea or deep depression of inspiratory motor output. The inhibitory effect could be correlated with the cooling temperature. Apnoeic response occurred either with complete absence of any inspiratory activity or combined with low level tonic inspiratory motor activity ('tonic apnoea'). The appearance of apnoea was CO2-independent, whereas the tonic component of the latter increased with increasing levels of PCO2. The results suggest that the structures in the deep, ventro-lateral aspect of rostral medulla, from which apnoea can be induced, correspond partly to the nucleus paragigantocellularis lateralis (nPGL) and the nucleus preolivaris. These structures appear to be relevant for the drive inputs necessary for respiratory rhythmogenesis. Unilateral focal cooling in the rostral medulla, including the 'B?tzinger Complex', caused increments in respiratory rate both in vagotomized and non-vagotomized animals. The increase in respiratory rate in response to cooling in the region of the 'B?tzinger Complex' was combined with either an enhancement or some depression of respiratory motor output. This area in the rostral part of the ventral respiratory group (VRG) seems not to be crucial for respiratory rhythmogenesis, but to play a role in determining both the intensity and timing of the respiratory activity. All effects of unilateral cold block were bilaterally symmetrical.     

Glycinergic inhibition is essential for co-ordinating cranial and spinal respiratory motor outputs in the neonatal rat

Eupnoeic breathing in mammals is dependent on the co-ordinated activity of cranial and spinal motor outputs to both ventilate the lungs and adjust respiratory airflow, which they do by regulating upper-airway resistance. We investigated the role of central glycinergic inhibition in the co-ordination of cranial and spinal respiratory motor outflows. We developed an arterially perfused neonatal rat preparation (postnatal age 0–4 days) to assess the effects of blocking glycine receptors with systemically administered strychnine (0.5–1 μM). We recorded respiratory neurones located within the ventrolateral medulla, inspiratory phrenic nerve activity (PNA) and recurrent laryngeal nerve activity (RLNA), as well as dynamic changes in laryngeal resistance. Central recordings of postinspiratory neurones revealed an earlier onset in firing relative to the onset of inspiratory PNA after exposure to strychnine (260 ± 38.9 vs. 129 ± 26.8 ms). After glycine receptor blockade, postinspiratory neurones discharged during the inspiratory phase. Strychnine also evoked a decrease in PNA frequency (from 38.6 ± 4.7 to 30.7 ± 2.8 bursts min⁻¹), but amplitude was unaffected. In control conditions, RLNA comprised inspiratory and postinspiratory discharges; the amplitude of the latter exceeded that of the former. However, after administration of strychnine, the amplitude of inspiratory-related discharge increased (+65.2 ± 15.2%) and exceeded postinspiratory activity. Functionally this change in RLNA caused a paradoxical, inspiratory-related glottal constriction during PNA. We conclude that during the first days of life in the rat, glycine receptors are essential for the formation of the eupnoeic-like breathing pattern as defined by the co-ordinated activity of cranial and spinal motor inspiratory and postinspiratory activities.     

Transgenic expression of fluorescent proteins in respiratory neurons

We screened transgenic mouse lines with Thy1.2 promoter-induced expression of fluorescent proteins (FPs) for targeting of respiratory neuronal populations in the medulla oblongata. Respiratory neurons were found to be tagged by FPs within the ventral respiratory column (VRC), the pre-B?tzinger complex (preB?tC) and the rostral ventral respiratory group (rVRG) interneurons. A subset of neurons in the preB?tC, labeled with the enhanced yellow fluorescent protein (EYFP), showed inspiratory activity during whole cell recordings from rhythmic slice preparations. Additionally, a subpopulation of EYFP-labeled preB?tC neurons expressed both NK1- and mu-opioid receptors. Furthermore, the spinal trigeminal nucleus, the lateral reticular nucleus (LRT) and the hypoglossal nucleus demonstrated intense EYFP expression whereas other regions of the medulla were devoid of neuronal EYFP labeling (e.g. the nucleus ambiguous). In conclusion, Thy1.2-FP transgenic mice will facilitate the functional analysis of respiratory-related neurons in the medulla and improve the three dimensional analysis of cells contributing to this important neuronal circuit.